The increasing prevalence of antibiotic resistance among clinically important pathogens is a growing threat, outpacing the development of antibiotics with new mechanisms of action. One novel approach that has drawn increasing attention is targeting bacterial virulence. One attractive class of targets is two component systems (TCSs), which sense changes in the environment and coordinate a cellular response. These bacterial targets are important during infection while absent in mammalian hosts. Moreover, TCSs are often conserved across species so that inhibitors would have the potential to be broad spectrum in activity. Pseudomonas aeruginosa, an important clinical pathogen with increasing antibiotic resistance, encodes one of the largest sets of TCSs known in bacteria and is thought to use these TCSs to coordinate the transition between growth in the external environment and in a human host, including the transition from acute to chronic infection lifestyles. It is becoming increasingly clear that TCS signaling is highly complex in P. aeruginosa. While canonical TCS signaling, defined as phosphorylation of a response regulator by its cognate histidine sensor kinase, is important, increasing evidence suggests that non-canonical TCS signaling may be equally important. Non-canonical signaling includes a number of different signaling mechanisms such as 1. physical interactions between sensors rather than phosphorylation events to modulate the activity of a response regulator, 2. signaling of a sensor through an alternative response regulator, or 3. signaling through multiple response regulators by one sensor kinase (cross-talk). We have identified several novel TCS not previously known to play a role in acute infection by screening a comprehensive set of P. aeruginosa mutants in 58 TCS sensor kinases in a new vertebrate model of acute infection. We identified kinB, phoR, bqsS, and copS as being required for full virulence in Danio rerio (zebrafish) embryos as well as gacS and retS, which have previously been shown to be required for infection in other models. We found that while KinB is required for acute infection, it signals non-canonically, independent both of its cognate response regulator, AlgB, and of its kinase activity. We propose to elucidate the mechanism of KinB's non-canonical TCS signaling and define the degree of non-canonical TCS signaling for the three other novel TCS (PhoR, BqsS, and CopS). Understanding the extent of non-canonical TCS signaling and the degree of cross-talk among TCS sensors and response regulators during acute vertebrate infection is critical for evaluating the potential of targeting TCS as a therapeutic strategy and will provide insight into the mechanisms that pathogens use to adapt to varying environments, including the human host to elicit disease. PUBLIC HEALTH RELEVANCE: Understanding how bacterial pathogens such as Pseudomonas aeruginosa sense and respond to their environments, including the human host, is critical to understanding how they adapt to the host microenvironment and cause disease. We have identified several Pseudomonas aeruginosa signaling systems called two component systems that are required to cause lethality in a vertebrate host model. We propose to elucidate the mechanisms by which they transduce these signals through intracellular response regulators; therefore, by understanding such molecular processes, we will evaluate the potential new paradigm of targeting such bacterial factors as a new therapeutic strategy in this current era of increasing antibiotic resistance.